PROJECT SUMMARY The activation of neurons in the brain leads to localized blood flow increases ? a phenomenon termed functional hyperemia. Widely used hemodynamic imaging techniques, such as fMRI, take advantage of functional hyperemia to infer neural activity from vascular responses. However, the vasculature seems to overcompensate in its reaction to neuronal activity ? blood flow increases over a larger region than the area of active neural tissue, and the increase in blood seems to exceed the oxygen needs of the tissue. Therefore, a deeper understanding of the degree to which blood flow changes reflect neural activity is critical for the accurate interpretation of hemodynamic imaging data. Additionally, although it is supposed that functional hyperemia is an efficient means of distributing limited resources, we know surprisingly little about how critical this blood flow increase is for the health and function of neural tissue. The overarching goal of this proposal is to understand the mechanisms and the functional role of the overshoot of blood supply in functional hyperemia. We recently found that individual vessels in the cortical parenchyma display stimulus-evoked blood flow increases even when the tissue around the vessel was unresponsive to the stimuli. In Aim 1, we will test if the increase in blood flow seen outside of the region of active neural tissue is caused by long-range propagation of arterial dilation signals through the pial network. Arterial dilation has been shown to propagate over long distances through endothelial cells in the vessel walls. We will modify a technique for disrupting this propagation using two-photon microscopy and determine if interrupting the propagation of vasodilation leads to a more precise correspondence between the locations of neural and vascular activity. In Aim 2, we will develop a technique for optically controlling the diameter of individual arterioles in vivo to study the effect of functional hyperemia on neural responses. Using two-photon optogenetics, we will prevent increased blood flow into regions of tissue which have been activated by sensory stimuli. We will analyze how the amplitude and stimulus selectivity of neuronal spiking and synaptic responses are affected by the lack of extra blood. These results will help us understand how normal neuronal function depends on robust neurovascular coupling. This in turn will shed light on whether the neurovascular coupling defects seen in many diseases are the cause of the accompanying neurological disorders. This proposal will help establish techniques and model systems for future studies aimed at understanding how neural activity leads to, and in turn depends on, local blood flow changes.